Neutralizing and protective murine monoclonal antibodies to the hemagglutinin of influenza H5 clades 2.3.2.1 and 2.3.4.4

Abstract Background Highly pathogenic avian H5 influenza viruses have spread and diversified genetically and antigenically into multiple clades and subclades. Most isolates of currently circulating H5 viruses are in clade 2.3.2.1 or 2.3.4.4. Methods Panels of murine monoclonal antibodies (mAbs) were generated to the influenza hemagglutinin (HA) of H5 viruses from the clade 2.3.2.1 H5N1 vaccine virus A/duck/Bangladesh/19097/2013 and the clade 2.3.4.4 H5N8 vaccine virus A/gyrfalcon/Washington/41088‐6/2014. Antibodies were selected and characterized for binding, neutralization, epitope recognition, cross‐reactivity with other H5 viruses, and the ability to provide protection in passive transfer experiments. Results All mAbs bound homologous HA in an ELISA format; mAbs 5C2 and 6H6 were broadly binding for other H5 HAs. Potently neutralizing mAbs were identified in each panel, and all neutralizing mAbs provided protection in passive transfer experiments in mice challenged with a homologous clade influenza virus. Cross‐reacting mAb 5C2 neutralized a wide variety of clade 2.3.2.1 viruses, as well as H5 viruses from other clades, and also provided protection against heterologous H5 clade influenza virus challenge. Epitope analysis indicated that the majority of mAbs recognized epitopes in the globular head of the HA. The mAb 5C2 appeared to recognize an epitope below the globular head but above the stalk region of HA. Conclusions The results suggested that these H5 mAbs would be useful for virus and vaccine characterization. The results confirmed the functional cross‐reactivity of mAb 5C2, which appears to bind a novel epitope, and suggest the therapeutic potential for H5 infections in humans with further development.


| INTRODUCTION
Since the first detection in 1997, highly pathogenic avian H5N1 influenza viruses have continued to spread and diversify both genetically and antigenically 1 with occasional human infection (https://cdn.who. int/media/docs/default-source/influenza/human-animal-interface-riskassessments/cumulative-number-of-confirmed-human-cases-for-avianinfluenza-a(h5n1)-reported-to-who--2003-2023.pdf?sfvrsn=a11e93cf_ 1&download=true). Genetic analysis has resulted in the classification of H5 virus isolates into multiple clades and subclades, 2 although most H5 viruses circulating in recent years have been in

| Cross-reactivity of mAb binding and neutralization
We investigated whether the two H5 mAb panels cross-reacted with the HAs of other H5 clades using ELISA binding and pseudovirus neutralization assays (  Figure 3A). The HA amino acid sequences of these vaccine viruses in the Sa and Sb antigenic regions are shown in Figure 3B. Only mAbs 3D6 and 5C2 were able to neutralize A/common magpie/   Figure S3) did not suggest an obvious explanation for the inability of 5C2 to neutralize the A/barn swallow virus. Epitope mapping by escape mutant isolation did not present a clear picture of the epitope recognized by 5C2. An S106R mutation from one escape mutant is not located on the outside of HA. A second escape mutant had two amino acid changes at K48E and K377T, the latter mutation also not located on the outside of HA. Nevertheless, the location of the K48E mutation, together with the results from competition experiments with head-and stem-binding mAbs, suggests that mAb 5C2 recognizes an epitope below the globular head but above the stalk region of HA. Previously described influenza stem-binding antibodies typically recognize many influenza subtypes, whereas the binding and neutralization properties of 5C2 appear broad, but only for other H5 viruses. Additional experimental approaches will be needed to more accurately define how 5C2 binds H5 influenza HA.
In summary, the murine mAbs described here should be useful reagents for characterization of influenza viruses with pandemic potential, as well as reagents for evaluating CVVs and candidate inactivated pandemic vaccines. For example, several previous studies have described the development of new assays that use mAbs for determining the potency of inactivated influenza vaccines. 10 The mAbs described here should be useful for setup of H5-specific potency assays. In particular, the cross-reactive mAb 5C2 may be an especially versatile antibody reagent. This mAb, or antibodies identified with similar specificities, may also have therapeutic potential for H5 infections in humans if developed as a humanized antibody.

| Cells and viruses
The H5 viruses used in these studies were reassortant CVVs (http:// www.who.int/influenza/vaccines/virus/en/). Influenza viruses were propagated in either 9-day-old specific pathogen-free embryonated chicken eggs or MDCK cells. Virus titer was determined by plaque assay on MDCK cells. All experiments, including animal experiments, were conducted at biosafety level 2 plus laboratory conditions.

| Monoclonal antibodies
mAbs to H5 HA were prepared as previously described. 11

| Pseudovirus neutralization assay
Pseudovirus production and neutralization assays were performed as previously described. 17

| Competition ELISA
Competition ELISA was set up essentially as described previously. 11 Briefly, ELISA plates were coated overnight with 2 μg/mL of soluble A/duck/Bangladesh/19097/2013 HA in PBS-based coating buffer (SeraCare, Gaithersburg, Maryland). Unlabeled mAbs were added to the wells in duplicate, starting at a concentration of 500 μg/mL, and were serially diluted on the plates and incubated for 2 h at 37 C. Purified mAb 5C2 was labeled with HRP using EZ-Link Activated Peroxidase Antibody Labeling Kit (ThermoFisher Scientific) and used as a detection antibody in competition with unlabeled mAbs at a concentration of 2 μg/mL for 1.5 h at 37 C. ABTS (SeraCare) was used as enzyme substrate.

| Selection and characterization of escape viruses
The selection of H5 escape virus mutants was performed in MDCK cells, essentially as described previously. 13 Approximately 10